Several approaches have been described to generate artificial oligomers with defined stoichiometries of recombinant antibodies or other proteins. This is desired, either because the binding affinity of a given protein to its target can be increased by multimerization, or because a certain biological effect can only be obtained with a certain oligomerization grade of the active compound (Crothers, D. M. & Metzger, H. (1972). The influence of polyvalency on the binding properties of antibodies. Immunochemistry, 9, 341-357). Another, newer review describes the effects of valency not only on functional affinity, but also on pharmacokinetics due to optimized molecular weight (Deyev S. M. & Lebedenko E. N. 2008, BioEssays 30:904-918).
Shu and coworkers have generated a variant of the GCN4 leucine zipper, that naturally is a dimer but in an engineered version forms a trimeric coil coil structure and used it to analyze the nature of trimeric assembly of the HIV-1 protein gp41 (Chemistry. 1999 Apr. 27; 38(17):5378-85).
Wyzgol and coworkers describe the usage of a trimerization domain from the chicken protein tenascin (amino acids 110-139, of UniProt entry: P10039. J Immunol 2009; 183:1851-1861), for the artificial trimerization of ligands of the TNF receptor superfamily. Biological activity could be strongly enhanced for some of their examples.
Secreted hemagglutinins with or without a carboxy-terminal trimerization domain based on the natural trimerization domain of T4 phage fibritin were expressed by Krammer and coworkers (PLOS ONE 2012, Volume 7, Issue 8, e43603). They demonstrated that the antigen was kept by this trimerization in a native like structure and was recognized by some epitope specific antibodies.
The first examples of dimeric or trimeric antibodies were the diabodies or triabodies (reviewed by Kortt and coworkers in Biomol Eng. 2001 Oct. 15; 18(3):95-108.). Those assemblies are essentially composed of scFv fragments where the interdomain linker is shortened such that the VH domain from one polypeptide cannot form a functional Fv with the VL domain from the same polypeptide, but form functional Fvs in an interchain manner.
An early review by Pluckthun and colleagues summarizes different approaches to generate dimeric or tetrameric scFv antibody fragment fusions (Immunotechnology 3 (1997) 83-105). Here the authors use the oligomerization domains that are either coiled-coil leucine zipper domains or are derived from the tetramerization domain of the human p53 tumor suppressor protein. Also a quantitative estimation is given on the enhancement of binding strength by multimerization of a given binding domain. For the purpose of generating a bivalent display of antibody fragments on a filamentous phage Lee and colleagues used a similar homodimerizing leucine zipper. Bivalent display was first achieved by the insertion of a dimerization domain, consisting of an IgG1 hinge region and a homodimerizing GCN4 leucine zipper, between a Fab and the C-terminal domain of the M13 gene-3 minor coat protein. Covalent linkage of the two zipper domains was obtained via the disulfide bonds coming from the hinge region, and display it on a phage for the screening of antibody libraries (J Immunol Methods. 2004 January; 284(1-2):119-32).
Cuesta and coworkers describe the usage of the Trimerization domains of either collagen XV or collagen XVIII for the generation of a trimeric scFv molecule, named trimerbody (2012, mAbs 4:2, 226-232). The trimerized constructs showed an almost 100-fold increase of the functional affinity compared to a monovalent scFv of the same specificity. Both trimerization domains form non-covalent trimers (Boudko et al.; J. Mol. Biol. (2009) 392, 787-802, and Wirz et al.; Matrix Biology 30 (2011) 9-15).
Binding of a ligand or antibody in a trimeric form to a certain receptor can have significant advantages over binding of monomeric or dimeric modules (like an IgG). Especially, binding to receptors of the TNFR family that induce apoptosis in the target cell after induced trimerization could generate a therapeutic benefit. Allen and coworkers generated a binder to Death receptor 4 based on a tetranectin C-type lectin domain (Mol Cancer Ther 2012; 11:2087-2095). This binding domain was trimerized via the coiled-coil motif obtained from the same tetranectin protein. This protein forms non-covalent homo-trimers. One of those trimeric molecules induced apoptosis in DR4 expressing cells, similar to the natural trimeric TRAIL ligand. This novel class of molecules was named atrimers by the authors (Mol Cancer Ther 2012; 11:2087-2095).
Fab fragments of an anti-ICAM-1 antibody were assembled into dimeric, trimeric or tetrameric format using the multimerization domain derived from either of the human transcription factors, ATFα or CREBPa (Charles et al.; Journal of Immunological Methods 284 (2004) 119-132). The oligomerization domains used here all share a coiled-coil motif, and assemble without the formation of covalent oligomers. These proteins successfully blocked rhinovirus infection in vitro, with the efficiency increasing from monomer to dimer, trimer, and tetramer.
So called “peptabodies” were generated by Terskikh and colleagues (Proc Natl Acad Sci USA. 1997 Mar. 4; 94(5):1663-8). A short peptide ligand was fused via a semi-rigid hinge region with the coiled-coil assembly domain of the cartilage oligomeric matrix protein (COMP), resulting in a pentameric multivalent binding molecule.
Heptamerization of binding domains from protein Z were generated via the heptamerization domain of the Archaeal RNA binding protein Sm1 through a flexible hinge peptide (Kim et al.; PLoS One. 2012; 7(8):e43077). Surface plasmon resonance (SPR) analysis showed that both heptameric anti-EGFR and anti-HER2 binders have a significantly enhanced binding strength to their target receptors with a nearly 100 to 1000 fold increase relative to the monomeric ligands.
Another approach to generate polyvalency was described by Li et al., and is named “chemically self-assembled antibody nanorings (CSANs)” (J. AM. CHEM. SOC. 2010, 132, 17247-17257). The authors designed it such that each nanoring subunit is composed of a number of artificial dimers of E. coli DHFR that assemble to the ring-like structures when a dimeric version of methotrexate (named: MTX2-C9) is added.
Bispecific antibodies present unique opportunities in terms of new applications for engineered antibodies. However, designing ideal bispecific antibodies remains a challenge.
Fick and coworkers give an overview on the generation of different bispecific reagents that comprise a scFv fragment fused to the trimerization domain of tenascin which is fused to a ligand of the TNF family (Patrick Chames (ed.), Antibody Engineering: Methods and Protocols, Second Edition, Methods in Molecular Biology, vol. 907, 597-609). Trimerization is stabilized by naturally occurring interchain disulfide bonds. Those molecules are useful for targeting approaches where the active, trimeric apoptosis inducer is delivered to a tumor cell via the anti-tumor binding capacity of the scFv fragment.
Stone and coworkers describe a novel bispecific antibody model in which five single domain antibodies (sdAbs) are fused via a linker sequence to the N-terminus of the verotoxin B (VTB) subunit, a pentamerization domain, and five sdAbs are fused via a linker sequence to the VTB C-terminus (Journal of Immunological Methods, (2007) 318 (1-2) pp. 88-94.). Several of such decavalent bispecific molecules, termed decabodies, were constructed and characterized. Albeit an interesting concept, the physico-chemical properties of those molecules are still to be optimized.
Kashentseva and coworkers use the fibritin domain of phage T4 to generate a bispecific, trimeric fusion protein that should redirect Adenoviruses to a tumor cell (Cancer Res Jan. 15, 2002 62; 609).
Provided herein are novel trimeric antigen binding molecules comprising a trimerisation domain derived from human cartilage matrix protein. Since the trimerization domain is derived from a protein of human origin the trimeric antigen binding molecule has a lower probability of immunogenicity compared to molecules with a polymerization domain of non-human origin. In addition, the trimerization domain derived from human cartilage matrix protein trimerizes into a coiled-coil structure through naturally occurring disulfide bonds which leads to a stable trimeric antigen binding molecule that can be used both in a monospecific and in a bispecific format.